Original Title: Regulatory GenomicsIn the present dissertation, I integrate contemporary high-throughput sequencing (HT-seq) methods to characterize regulatory elements and transcriptional units in several biological systems in order to understand the molecular networks regulating the processes of gene expression. Our intense efforts contributed to several discoveries in biology, including a. the discovery of novel small RNA species that regulate mRNAs stability - the riRNAs, b. finding novel transcriptional units that regulate estrogen-dependent transcriptional events - the eRNAs, c. establishing the role of long noncoding RNAs in androgen-dependent gene expression - the lincRNAs PCGEM1 and PRNCR1, d. elucidating a novel epigenomics regulator - PHF8 as a H4K20me1 demethylase, e. characterizing a multifaceted transcrip- tional regulator - GPS2, f. identifying novel GWAS loci located in enhancers that carry disease susceptibility alleles. Chapter 1 reviews the current knowledge on genome regulatory elements, transcriptional units and chromatin architecture, and it describes 8 computational protocols that I have established in the laboratory for data analysis, together with their biological applications and methodological limitations. Chapter 2 details the use of HT-seq methods to characterize 2 novel transcriptional regulators (PHF8 and GPS2 ) that associate with promoters. Chapter 3 describes a novel small RNA species (riRNAs ), that derives from Alu retrotransposons and was bioin- formatically predicted to target the mRNAs. Chapter 4 describes the functions of enhancer RNAs in gene expression and chromatin interactions, by employ- ing GRO-seq and 3D-DSL methods. Chapter 5 describes 2 lincRNAs (PRNCR1, PCGEM1 ) that are over-expressed in prostate cancers and regulate tumor growth, by using ChIRP-seq and GRO-seq methods. Chapter 6 describes the biological functions of an enhancer in 9p21 gene desert, that carries genetic susceptibility to disease, and advances in 3D-DSL technology. I conclude with a section that summarizes these discoveries and outlines the potential areas of future research.